Chronic excessive alcohol consumption causes endothelial dysfunction (ED), which can predispose alcoholics to cardiovascular diseases. Long-term withdrawal of alcohol unfortunately cannot correct impaired vascular function, highlighting the need to understand its pathogenesis and to seek new therapeutic strategies for alcohol- induced vascular injury. Although the sustained oxidative stress induced by ethanol metabolism is a dominant driving force for ED, the underlying redox mechanisms remain unclear, and current antioxidant therapies have very limited cardiovascular benefits, partially due to the ineffective removal of extremely short-lived reactive oxygen species (ROS) and the resultant oxidative damages accumulated chronically. In this application, we will explore whether S-glutathionylation (PrS-SG), a stable and revesible oxidant-induced posttranslational modification of protein cysteine thiol groups, could be a better target over ROS for interventions for alcohol- induced ED, because it mediates the cellular action of ROS, and more importantly, this stable modification can be removed by de-glutathionylation enzymes, such as glutaredoxin1 (Grx1), which appears to be critical for cellular responses to oxidative stress. In endothelial cells (ECs), chronic ethanol exposure selectively increases the level of PrS-SG. However, whether and how Grx1/PrS-SG axis is involved in alcohol-induced ED is utterly unknown. Our preliminary studies show that in human aortic ECs (HAEC), ethanol causes a concurrent decrease in Grx1 protein and accumulation of PrS-SG in a dose-dependent manner. Increasing Grx1 expression promotes EC barrier function and NO biogenesis. These exciting findings lead to our central hypothesis that Grx1, as a novel endothelial cell protector, prevents and reverses chronic alcohol-induced vascular dys- function by maintaining redox homeostasis. To test this hypothesis, we will: (1) define the relationship between aortic Grx1/PrS-SG and alcohol-induced ED; and (2) elucidate the redox mechanisms for the protective effect of Grx1 on chronic alcohol-induced ED. Under Aim 1, a mouse model of chronic-plus-binge ethanol feeding that closely resembles the heavy drinking patterns in humans and developed by NIAAA investigators will be used to characterize the changes in aortic Grx1/PrS-SG and the onset and progression of alcohol-induced ED. A novel, inducible, endothelial-specific Grx1 transgenic (TG) mice will be used to test whether endothelial Grx1 can protect and reverse PrS-SG and ED caused by chronic ethanol feeding. Under Aim 2, using the endothelial- specific Grx1 TG mice and isolated aortic endothelial cells from Grx1 TG and knockout mice, we will focus on studying the impact of Grx1 on the integrated redox signaling of small RhoGTPase Rac1 and NAD+-dependent deacetylase SIRT1, which both converge to mediate cellular responses to ethanol. The outcomes of the proposed research will help advance our understanding of how chronic alcohol abuse damages vascular endo- thelial cells, and offer a novel therapeutic strategy to remove pathogenic protein cysteine thiol oxidation, thereby repairing vascular injury and reducing the burden of cardiovascular diseases in chronic alcoholics.